Development of HPLC-MS/MS method for the simultaneous determination of environmental phenols in human urine

We present a sensitive method for simultaneous determination of bisphenol A (BPA), benzophenone-3 (BP-3), 4-tert-octylphenol (t-OP), ortho-phenylphenol (OPP), four parabens (methyl, ethyl, propyl, butyl parabens) and five chlorophenols (2,4-dichlorophenol (2,4-DCP), 2,5-dichlorophenol (2,5-DCP), 2,4,5-trichlorophenol (2,4,5-triclorophenol), 2,4,6-trichlorophenol (2,4,6-TCP), and triclosan (TCS)), in human urine by high-pressure liquid chromatography (HPLC) mass spectrometry (MS). Samples were processed using enzymatic deconjugation of glucuronides followed by solid phase extraction (SPE) on a C18 cartridge and the eluate was concentrated. Analytes were separated by reversed-phase HPLC and then detected by atmospheric pressure chemical ionisation (APCI) MS and quantified by isotope dilution method. We describe details for optimisation of each step of the procedure. The sample treatment steps are straightforward and not labour-intensive and, therefore, permit a high sample throughput with excellent prospects for automation. This method shows low inter-day variation, and detection limits for most of the compounds are below 1 ng/mL in 1 mL of urine. The method accuracy was also verified by the analysis of proficiency testing urine samples.     

固相萃取-气相色谱-质谱法测定木制家具中氯酚类及菊酯类防腐剂

建立了固相萃取-气相色谱-质谱(SPE-GC-MS)检测含氯酚类化合物(2,4-二氯苯酚、2,4,6-三氯苯酚、2,4,5-三氯苯酚、2,3,4,6-四氯苯酚、五氯苯酚、林丹)和菊酯类化合物(氯菊酯、氟氯氰菊酯、氯氰菊酯、溴氰菊酯)等10种木材防腐剂的方法。对家具样品采用超声萃取法、以甲醇为提取剂在室温下反复提取2次,提取液经浓缩后,加入碳酸钾和乙酰酐衍生化,将衍生化后的溶液通过Oasis HLB固相萃取柱净化,用乙酸乙酯洗脱并收集检测。采用该方法实现了家具中10种木材防腐剂的分离检测,该方法中氯酚类防腐剂定量限为1 mg/kg、菊酯类防腐剂定量限为5 mg/kg,平均回收率为76.0%～108.8%。应用该方法对市场上销售的木制家具进行了检测,在部分家具中检出含有少量林丹。实验结果证明,该方法准确、灵敏,可有效地应用于木制家具中防腐剂的实际检验工作中。     

Sensitivity enhancement of chlorinated phenols by continuous flow liquid membrane extraction followed by capillary electrophoresis

This paper describes a new analytical system, based on the combination of continuous flow liquid membrane extraction (CFLME) enrichment and capillary electrophoresis (CE) separation, for analysis of chlorinated phenols in water samples. Five chlorinated phenols including 3-chlorophenol (3CP), 4-chlorophenol (4CP) 2,4-dichlorophenol (DCP), 2,4,6-trichlorophenol (TCP), and pentachlorophenol (PCP) were separated by CE with Tris/sodium dihydrogen phosphate solution containing methanol 1% (v/v) as the run buffer. CFLME related parameters were investigated and optimal enrichment was obtained by using 0.3 mol L(-1) Tris as acceptor and with a sample pH 5.0, a sample flow rate of 4.0 mL min(-1), and an enrichment sample volume of 150 mL. The detection limit (S/N= 3) was 6.9, 1.0, and 1.7 ng mL(-1) for DCP, PCP, and TCP, respectively. The reproducibility (RSD%, n = 6) was 5.7 for DCP, 2.5 for PCP, and 2.8% for TCP (n = 6). The proposed method was applied to the determination of chlorinated phenols in spiked water samples with relatively satisfactory recoveries.     

Automated on-line column-switching HPLC-MS/MS method for measuring environmental phenols and parabens in serum

We developed a method using on-line solid phase extraction (SPE) coupled to high performance liquid chromatography–isotope dilution tandem mass spectrometry (HPLC–MS/MS) to measure the serum concentrations of seven environmental phenols and five parabens: bisphenol A; ortho-phenylphenol; 2,4-dichlorophenol; 2,5-dichlorophenol; 2,4,5-trichlorophenol; benzophenone-3; triclosan; and methyl-, ethyl-, propyl-, butyl-, and benzyl-parabens. The phenols and parabens present in serum were retained and concentrated on a C18 reversed-phase size-exclusion SPE column, back-eluted from the SPE column while the eluate was diluted through a mixing Tee (analyte peak focusing), separated using a pair of monolithic HPLC columns, and detected by isotope dilution-MS/MS. Sample preparation did not require protein precipitation, only dilution of the serum with 0.1 M formic acid. This method, which combines an on-line SPE with analyte peak focusing feature and the selective atmospheric pressure photoionization MS detection, resulted in limits of detection ranging from 0.1 to 0.5 ng/mL for most of the analytes. The high throughput and adequate sensitivity with yet a relative low serum volume used (100 μL) confirm that analytically it is possible to measure simultaneously these phenols and parabens with the precision and accuracy at sub-parts-per-billion levels required for biomonitoring. However, important additional factors, including validated sample collecting, handling, and storing protocols, as well as toxicokinetic data, are required if these measures are used for exposure assessment.     

A comparison between positive and negative ion modes in thermospray liquid chromatography-mass spectrometry for the determination of chlorophenols and herbicides

Summary Positive and negative ion modes (Pl and NI, respectively) have been employed for the characterization of 2,4-dichlorophenol, 2,4,5-trichlorophenol, pentachlorophenol, Linuron and Cyanazine in thermospray (TSP) liquid chromatography-mass spectrometry (LC-MS). The PI mode showed no response when 200 ng of the different chlorophenols were injected, while for Linuron and Cyanazine high signals were obtained with [M+NH₄]⁺ and [M+acetic acid]⁺ ions as base peaks, respectively. With the NI mode, the base peak usually corresponds to the [M−H]⁻ ion, with better sensitivities for the chlorophenols than for the herbicides. The chloride adduct [M+Cl]⁻ ion was also obtained for 2,4,5-trichlorophenol and for Linuron. Although the PI mode is more sensitive than the NI mode for the two herbicides, the combination of both ionization modes offers complementary structural information for characterizing such compounds in TSP LC-MS.     

An in‐line SPE strategy to enhance sensitivity in CE for the determination of pharmaceutical compounds in river water samples

In this study, the suitability of solid‐phase extraction (SPE) coupled in‐line to CE with UV–Vis detection was evaluated for the preconcentration and separation of diluted solutions of five pharmaceuticals compounds: benzafibrate, piroxicam, diclofenac sodium, naproxen and clofibric acid. An SPE analyte concentrator containing Oasis^® HLB sorbent was constructed without frits and placed near the inlet end of the separation capillary. Different parameters such as sample pH, composition and volume of the elution plug and sample loading time were studied in order to obtain the maximum preconcentration factors. The LODs reached for standard samples were in the range 0.06–0.5 ng/mL with good reproducibility, and the developed strategy provides sensitivity enhancement factors around 14 000‐fold in peak area and 5900‐fold in peak height compared with the normal hydrodynamic injection. Finally, river water samples fortified with the pharmaceutical compounds were analyzed by the developed in‐line SPE‐CE‐UV method in order to show the potential of the methodology for the analysis of environmental aquatic samples. For these samples, high values of relative recoveries, between 73–107% and 79–103% for two concentration levels, 5 and 25 ng/mL, respectively, were obtained and LODs ranged between 0.19 and 1 ng/mL.     

Evaluation of liquid-phase microextraction conditions for determination of chlorophenols in environmental samples using gas chromatography-mass spectrometry without derivatization

Determination of trace chlorophenols (CPs) in environmental samples has been evaluated using liquid-phase microextraction (LPME) coupled with gas chromatography-mass spectrometry (GC-MS) without derivatization. The LPME procedure used to extract CPs from water involved 15 microL 1-octanol as acceptor solution in a 5.0 cm polypropylene hollow fiber with an inner diameter of 600 microm and a pore size of 0.2 microm. Under the optimal extraction conditions, enrichment factors from 117 to 220 are obtained. The obtained linear range is 1-100 ng mL(-1) with r(2)=0.9967 for 2,4-dichlorophenol (2,4-DCP); 1-100 ng mL(-1) with r(2)=0.9905 for 2,4,6-trichlorophenol (2,4,6-TCP); 5-500 ng mL(-1) with r(2)=0.9983 for 2,3,4,6-tetrachlorophenol (2,3,4,6-TeCP), and 10-1000 ng mL(-1) with r(2)=0.9929 for pentachlorophenol (PCP). The limits of detection range from 0.08 to 2 ng mL(-1), which is comparable with the reported values (12-120 ng mL(-1)). Recoveries of CPs in various matrices exceed 85% with relative standard deviations of less than 10%, except for PCP in landfill leachate. The applicability of this method was examined to determine CPs in environmental samples by analyzing landfill leachate, ground water and soil. The 2,4-DCP and 2,4,6-TCP detected in the landfill leachate are 6.68 and 2.47 ng mL(-1). The 2,4,6-TCP detected in ground water is 2.08 ng mL(-1). All the studied CPs are detected in contaminated soil. The proposed method is simple, low-cost, less organic solvent used and can potentially be applied to analyze CPs in complex environmental matrices.     

Determination of UV filters in river water samples by in‐line SPE‐CE‐MS

The use of SPE coupled in‐line to CE using electrospray MS detection (in‐line SPE‐CE‐ESI‐MS) was investigated for the preconcentration and separation of four UV filters: benzophenone‐3, 2,2‐dihydroxy‐4‐methoxybenzophenone, 2,4‐dihydroxybenzophenone and 2‐phenylbenzimidazole‐5‐sulphonic acid. First, a CE‐ESI‐MS method was developed and validated using standard samples, obtaining LODs between 0.06 μg/mL and 0.40 μg/mL. For the in‐line SPE‐CE‐ESI‐MS method, three different sorbents were evaluated and compared: Oasis HLB, Oasis MCX, and Oasis MAX. For each sorbent, the main parameters affecting the preconcentration performance, such as sample pH, volume, and composition of the elution plug, and sample injection time were studied. The Oasis MCX sorbent showed the best performance and was used to validate the in‐line SPE‐CE‐ESI‐MS methodology. The LODs reached for standard samples were in the range between 0.01 and 0.05 ng/mL with good reproducibility and the developed strategy provided sensitivity enhancement factors between 3400‐fold and 34 000‐fold. The applicability of the developed methodology was demonstrated by the analysis of UV filters in river water samples.     

Evaluation of fritless solid‐phase extraction coupled on‐line with capillary electrophoresis‐mass spectrometry for the analysis of opioid peptides in cerebrospinal fluid

Fritless SPE on‐line coupled to CE with UV and MS detection (SPE‐CE‐UV and SPE‐CE‐MS) was evaluated for the analysis of opioid peptides. A microcartridge of 150 μm id was packed with a C₁₈ sorbent (particle size > 50 μm), which was retained between a short inlet capillary and a separation capillary (50 μm id). Several experimental parameters were optimized by SPE‐CE‐UV using solutions of dynorphin A (DynA), endomorphin 1 (End1), and methionine‐enkephaline (Met). A microcartridge length of 4 mm was selected, sample was loaded for 10 min at 930 mbar and the retained peptides were eluted with 67 nL of an acidic hydro‐organic solution. Using SPE‐CE‐MS, peak area and migration time repeatabilities for the three opioid peptides were 12–27% and 4–5%, respectively. SPE recovery was lower for the less hydrophobic DynA (22%) than for End1 (66%) and Met (78%) and linearity was satisfactory in all cases between 5 and 60 ng/mL. The LODs varied between 0.5 and 1.0 ng/mL which represent an enhancement of two orders of magnitude when compared with CE‐MS. Cerebrospinal fluid (CSF) samples spiked with the opioid peptides were analyzed to demonstrate the applicability to biological samples. Peak area and migration time repeatabilities were similar to the standard solutions and the opioid peptides could be detected down to 1.0 ng/mL.     

Comparison of two different derivatization procedures for the determination of urinary chlorophenol excretions

A GC/MS method with enhanced sensitivity and specificity suitable for the determination of various chlorophenols in the urine of persons occupationally and environmentally exposed to diverse chlorinated aromatic hydrocarbons has been elaborated and compared with the method customarily used. After acid hydrolysis and steam distillation, followed by several clean-up steps, the chlorophenols have been derivatized with N-heptafluorobutyrylimidazole (HFBI), yielding ester derivatives. This procedure facilitates the detection of 3-and 4-monochlorophenol, 2,4- and 2,5-dichlorophenol, 2,4,5-and 2,4,6-trichlorophenol as well as 2,3,4,6-tetrachlorophenol down to 0.1–1 g/l, depending on the number of chlorine atoms. A comparison to the commonly used derivatization procedure with diazomethane revealed, except for 2,4,5-trichlorophenol, satisfactory conformity of both techniques. Due to an improvement of the detection limit by a factor of five for the monochlorophenols, for the first time the quantification of 3-monochlorophenol and the identification of 2-monochlorophenol as constituents of human urine have been possible as HFBI-derivatives. The excretion of mono-, di-, tri- and tetrachlorophenols in the urine of the general population could be confirmed.     

Aquatic degradation of triclosan and formation of toxic chlorophenols in presence of low concentrations of free chlorine

The degradation of 2-(2,4-dichlorophenoxy)-5-chlorophenol (triclosan) in chlorinated water samples was investigated. Sensitive determination of the parent compound and its transformation products was achieved by gas chromatography with mass spectrometry detection after sample concentration, using a solid-phase extraction sorbent and silylation of the target compounds. Experiments were accomplished using ultrapure water spiked with chlorine and triclosan concentrations in the low mg/l and ng/ml ranges respectively. Chlorination of the phenolic ring and cleavage of the ether bond were identified as the main triclosan degradation pathways. Both processes led to the production of two tetra- and a penta-chlorinated hydroxylated diphenyl ether, as well as 2,4-dichlorophenol. The formation of 2,3,4-trichlorophenol was not detected in any experiment; however, significant amounts of 2,4,6-trichlorophenol were noticed. All of these five compounds were also identified when triclosan was added to tap-water samples with free chlorine concentrations below 1 mg/l. Minor amounts of three di-hydroxylated phenols, containing from one to three atoms of chlorine in their structures, were also identified as unstable triclosan chlorination by-products. The analysis of several raw wastewater samples showed the co-existence of important concentrations of triclosan and its most stable by-products (2,4-dichlorophenol and 2,4,6-trichlorophenol), reinforcing the potential occurrence of the described transformations when products containing triclosan are mixed with chlorinated tap water.     

Derivative spectrophotometry in the analysis of mixtures of phenols and herbicides

Derivative spectrophotometry (zero-crossing technique) was applied to the determination of selected phenols and herbicides in two-component mixtures. Methyl- and chlorophenols (3-methylphenol, 2,3- and 3,4-dimethylphenol, 2,5-, 2,6- and 3,4-dichlorophenol and 2,4,5-trichlorophenol) and triazine, uracil and urea herbicides (simazine, propazine, hexazinone, bromacil and metoxuron) were examined. The RSD values ranged between 0.05 and 4% and the recoveries obtained were between 97 and 110%. The developed derivative spectrophotometric method was also applied as a complementary technique for the separation of overlapping peaks of sample compounds obtained by HPLC with diode-array detection. Metoxuron and 3-methylphenol, metoxuron and 2,5-dichlorophenol and simazine and 2,6-dichlorophenol were determined simultaneously by this method at the level of 1 x 10(-3) g l-1.     

Programed nebulizing-gas pressure mode for quantitative capillary electrophoresis-mass spectrometry analysis of endocrine disruptors in honey

The application of programed nebulizing-gas pressure (PNP) has been previously described to be a simple strategy for the separation of anions by capillary electrophoresis-electrospray-mass spectrometry (CE-ESI-MS). The PNP mode provided high resolution and stable analyses and also had the advantage of allowing the use of capillaries wider than the 50-75 μm conventional ones. Here, the application of the PNP approach to the quantitative analysis of pollutants in real samples by CE-ESI-MS is described for the first time; in particular, for the determination several endocrine disruptors (2,4-dichlorophenol, 2,4,5-trichlorophenol, pentachlorophenol, bisphenol-A, 4-tert-butyl-phenol, and 4-tert-butyl benzoic acid) in honey. For sample pretreatment, different liquid-liquid extraction (LLE) procedures were assayed and compared to the QuEChERS(?) methodology prior to electrophoretic analysis. With the application of the PNP approach to CE-ESI-MS, the limits of detection achieved were in the 1-4 ng/g range with a simple liquid-liquid procedure without any further clean-up step; relative standard deviation values in the 2-9% range were found. The analytical characteristics allow the proposed method to be used in the control analysis of these compounds in honey.     

Detection and determination of chlorophenols and chlorophenoxyacetic acids by instrumental analysis

A quantitative method for the determination of chlorophenols and chlorophenoxyacetic acids in aqueous solutions is described. The samples investigated contained 2-chlorophenol, 2,4-dichlorophenol, 2,6-dichlorophenol, 2,4,6-trichlorophenol and their phenoxyacetic acid derivatives. The total amount of chlorophenols is determined by spectrophotometry, the ratio of individual chlorophenols by gas chromatography and the total quantity of phenoxyacetic acids by acidimetric titration. The determinations are carried out after extraction with diethyl ether, carbon tetrachloride and petroleum ether, respectively.     

A Method for Determination of Low Levels of Exposure to 2,4-D and 2,4,5-T

A method has been developed for the determination of trace quantities of 2,4-dichloro-phenoxyacetic acid (2,4-D), 2,4,5-trichlorophenoxyacetic acid (2,4,5-T), 2,4-dichlorophenol (2,4-DCP), and 2,4,5-trichlorophenol (2,4,5-TCP) in human and rat urine. The method involves acid hydrolysis of the phenolic conjugates, extraction of the free phenols and acids, ethylation with diazoethane, silica-gel column chromatography clean-up of the derivatized urine extract, and gas chromatographic determination using the electron-capture detector. The average recoveries of 2,4-D, 2,4,5-T, 2,4-DCP, and 2,4,5-TCP from rat urine spiked with known amounts of the herbicides and their phenols were 94%, 98%, 92%, and 90%, respectively. The limits of detection for 2,4-D, 2,4,5-T, DCP, and TCP in rat urine were: 0.05, 0.01, 0.10, and 0.01 ppm, respectively. The method was used to analyze urine of rats given various levels of 2,4-D and 2,4,5-T by gavage. Results showed that levels of exposure of 3.75 mcg/kg for 2,4-D and 5.0 mcg/kg for 2,4,5-T in rats can be detected in urine within 24 hr from exposure. Urine samples from occupationally exposed people were analyzed and found to contain 0.2 to 1.0 ppm 2,4-D and 0.05 to 3.6 ppm 2,4,5-T.     

Comparative studies on the oxidative dechlorination of chlorophenols by a superoxide complex

Transition Metal Chemistry - The chlorophenols (CPs), 4-chlorophenol (4-CP), 2,4-dichlorophenol (2,4-DCP) and 2,4,6-trichlorophenol (2,4,6-TCP), are potent environmental hazards. They can be...     

Purge-assisted headspace solid-phase microextraction combined with gas chromatography-mass spectrometry for determination of chlorophenols in aqueous samples

A simple, economical and very effective method is demonstrated for simultaneous determination of 2,4-dichlorophenol, 2,4,6-trichlorophenol, 2,3,4,6-tetrachlorophenol and pentachlorophenol, in aqueous samples, by using purge-assisted headspace solid-phase microextraction (PA/HS-SPME) coupled to gas chromatography-mass spectrometry (GC-MS). In the new method, purging the sample enhances the removal of the trace chlorophenols without derivatization from the matrices to the headspace. Extraction parameters including extraction temperature, purge gas flow rate and extraction time were systematically investigated. Under optimal conditions, the relative standard deviations (RSDs) were 4-11% at 50 pg/mL and 5-14% at 5 pg/mL, respectively. The recoveries were in the range of 83-114%. Detection limits were determined at the fg level. These results indicate that PA/HS-SPME provides a significant contribution to highly efficient extraction of semi-volatile CPs, especially for pentachlorophenol, which has the smallest Henry's constant and large octanol-water partitioning coefficient. In addition, the proposed method was successfully applied to the analysis of chlorophenols in landfill leachate. New perspectives are opened for headspace extraction of relatively low vapor pressure compounds in complex matrices.     

改进的五氟苄基溴衍生化反应测定水中酚类化合物

建立了气相色谱-质谱法测定水中6种酚类化合物(2,6-二氯酚、2,4-二氯酚、2,4,6-三氯酚、2,4,5-三氯酚、2,3,4,6-四氯酚和五氯酚)的方法.样品经二氯甲烷-乙酸乙酯混合溶剂萃取后,用旋转蒸发浓缩至1 mL,加入五氟苄基溴进行改进版衍生化反应,产物用DB-5 mS毛细管柱分离,采用选择离子监测模式测定....     

Synthesis of porous divinylbiphenyl copolymers and their sorptive properties towards phenol and its derivatives

Seven porous divinylbiphenyl polymers having the same nominal crosslinking degree (51.8 wt.%) have been synthesized using suspension polymerization method in the presence of the following inert diluents or their mixtures: toluene, heptane, dodecane, isooctane. The use of various inert diluents was aimed at changing the extent of polymeric network-diluent interactions. The obtained polymers have specific surface area in the range 50-300 m²/g depending on the type and amount of inert diluents used during polymerization. Their sorptive properties have been studied using dilute (0.5 mmol/l) aqueous solutions of phenol and its derivatives (2-chlorophenol, 2,4-dichlorophenol, 2,4,5-trichlorophenol, 2,6-dimethylphenol, 4-hydroxyphenol). It has been found that sorption, at low equilibrium concentration, follows the order: 2,4,5-trichlorophenol > 2,4-dichlorophenol > 2-chlorophenol > 2,6-dimethylphenol > phenol > 4-hydroxyphenol. Full characteristic of the porous structure of polymers has been obtained by nitrogen adsorption at 77K.     

Development of a static and exhaustive extraction procedure for field passive preconcentration of chlorophenols in environmental waters with hollow fiber-supported liquid membrane

A static and exhaustive extraction mode of hollow fiber-supported liquid membrane was developed for field sample passive pretreatment of environmental water samples. The extraction device was prepared by immobilizing dihexyl ether in the wall of a polypropylene hollow fiber membrane (60 cm length, 50 μm wall thickness, and 280 μm id) as liquid membrane and filling the fiber lumen with 0.1 M NaOH as acceptor, and closing the two ends of the fiber with an aluminum foil. Passive extraction was conducted by immersing the device into 15 mL water samples modified with 0.01 M HCl and 20% m/v NaCl. Model analytes including 4-chlorophenol, 2,4-dichlorophenol, and 2,4,6-trichlorophenol were transferred into acceptor with extraction efficiencies over 79% in 10 h at room temperature, and determined by high-performance liquid chromatography. The proposed method has the enrichment factor of 394-498 and LOD of 0.3-0.4 μg/L for the three chlorophenols. Humic acid and salinity in the environmentally relevant range had no significant influence on the extraction, and chlorophenols in various environmental waters were determined with spike recoveries between 71.6 and 120%. The static passive extraction nature benefited field sample pretreatment without power, whereas the exhaustive extraction mode effectively eliminated the sample matrix effects.